Compositions and methods to detect Candida albicans nucleic acid

ABSTRACT

Compositions are disclosed as nucleic acid sequences that may be used as amplification oligomers, including primers, capture probes for sample preparation, and detection probes specific for  Candida albicans  26S rRNA sequences or DNA encoding 26S rRNA. Methods are disclosed for detecting the presence of  C. albicans  in samples by using the disclosed compositions in methods that include in vitro nucleic acid amplification of a 26S rRNA sequence or DNA encoding the 26S rRNA sequence to produce a detectable amplification product.

FIELD OF THE INVENTION

This invention relates to detection of the presence of fungi in a sampleby using molecular biological methods, and specifically relates todetection of Candida albicans in a sample by amplifying C. albicansnucleic acid sequences and detecting the amplified nucleic acidsequences.

SUMMARY

Disclosed are methods of detecting Candida albicans in a sample,including environmental samples, biopharmaceutical samples andbiological specimens derived from infected humans, by amplifying anddetecting target sequences contained in C. albicans 26S rRNA or DNAencoding 26S rRNA. By using specific amplification and detection probeoligomers disclosed herein, the methods amplify target sequences in 26SrRNA sequences of C. albicans and detect the amplified products. Someembodiments monitor the development of specific amplification productsduring the amplification step. Some embodiments include a sampleprocessing step using a capture probe oligomer.

A method is disclosed for detecting Candida albicans in a sample,comprising the steps of: mixing a sample that contains a C. albicanstarget nucleic acid that is a 26S rRNA sequence or DNA encoding the 26SrRNA sequence with a first amplification oligomer comprising a targetbinding region consisting of SEQ ID NO: 1 or SEQ ID NO: 2 and a secondamplification oligomer comprising a target binding region consisting ofSEQ ID NO: 6; providing an enzyme with nucleic acid polymerase activityand nucleic acid precursors to make an amplification mixture thatincludes the first and second amplification oligomers and the C.albicans target nucleic acid; elongating in vitro a 3′ end of at leastone of the amplification oligomers hybridized to the C. albicans targetnucleic acid by using the enzyme with nucleic acid polymerase activityand the C. albicans target nucleic acid as a template to produce anamplified product; and, detecting the amplified product by hybridizingthe amplified product specifically to a detection probe oligomercomprising a target binding sequence consisting of SEQ ID NO:10 toindicate the presence Candida albicans in the sample. Some embodimentsalso include a sample processing step that captures the C. albicanstarget nucleic acid from the sample before the mixing step. The sampleprocessing step may use a capture probe oligomer that contains a targetbinding sequence consisting of SEQ ID NO: 35 or 36, wherein the targetbinding sequence is optionally covalently attached to a 3′ tailsequence.

A composition is disclosed for detecting Candida albicans 26S rRNAsequence or DNA encoding the 26S rRNA sequence by using in vitroamplification, comprising a first amplification oligomer comprising atarget binding region consisting of SEQ ID NO: 1 or SEQ ID NO: 2, asecond amplification oligomer comprising a target binding regionconsisting of SEQ ID NO: 6, and a detection probe oligomer comprising atarget binding region consisting of SEQ ID NO: 10. Some embodiments alsoinclude at least one capture probe oligomer that contains a targetbinding region consisting of SEQ ID NO: 35 or 36, optionally with a 3′tail sequence covalently attached to the target binding sequence.

Another method is disclosed for detecting Candida albicans in a sample,comprising the steps of: mixing a sample that contains a C. albicanstarget nucleic acid that is a 26S rRNA sequence or DNA encoding the 26SrRNA sequence with a first amplification oligomer comprising a targetbinding region consisting of SEQ ID NO: 19 and a second amplificationoligomer comprising a target binding region consisting of SEQ ID NO: 24;providing an enzyme with nucleic acid polymerase activity and nucleicacid precursors to make an amplification mixture that includes the firstand second amplification oligomers and the C. albicans target nucleicacid; elongating in vitro a 3′ end of at least one of the amplificationoligomers hybridized to the C. albicans target nucleic acid by using theenzyme with nucleic acid polymerase activity and the C. albicans targetnucleic acid as a template to produce an amplified product; and,detecting the amplified product by hybridizing the amplified productspecifically to a detection probe oligomer comprising a target bindingsequence consisting of SEQ ID NO:28 to indicate the presence Candidaalbicans in the sample. Some embodiments also include a sampleprocessing step that captures the C. albicans target nucleic acid fromthe sample before the mixing step. The sample processing step may use acapture probe oligomer that contains a target binding sequenceconsisting of SEQ ID NO: 35 or 36, wherein the target binding sequenceis optionally covalently attached to a 3′ tail sequence.

Another composition is disclosed for detecting Candida albicans 26S rRNAsequence or DNA encoding the 26S rRNA sequence by using in vitroamplification, comprising a first amplification oligomer comprising atarget binding region consisting of SEQ ID NO: 19, a secondamplification oligomer comprising a target binding region consisting ofSEQ ID NO: 24, and a detection probe oligomer comprising a targetbinding region consisting of SEQ ID NO: 28. Some embodiments alsoinclude at least one capture probe oligomer that contains a targetbinding region consisting of SEQ ID NO: 35 or 36, optionally with a 3′tail sequence covalently attached to the target binding sequence.

Other methods and compositions are disclosed for amplifying anddetecting Candida albicans 26S rRNA sequence or DNA encoding the 26SrRNA sequence. The methods and compositions may comprise oligomercombinations of target binding regions for a first amplificationoligomer, a second amplification oligomer, and a detection probeoligomer, respectively, as follows: SEQ ID NOs: 1, 4 & 10; SEQ ID NOs:1, 4 & 12; SEQ ID NOs: 1, 4 & 14; SEQ ID NOs: 2, 4 & 10; SEQ ID NOs: 2,4 & 12; SEQ ID NOs: 2, 4 & 14; SEQ ID NOs: 3, 4 & 10; SEQ ID NOs: 3, 4 &12; SEQ ID NOs: 3, 4 & 14; SEQ ID NOs: 1, 6 & 10; SEQ ID NOs: 1, 6 & 12;SEQ ID NOs: 1, 6 & 14; SEQ ID NOs: 2, 6 & 10; SEQ ID NOs: 2, 6 & 12; SEQID NOs: 2, 6 & 14; SEQ ID NOs: 3, 6 & 10; SEQ ID NOs: 3, 6 & 12; SEQ IDNOs: 3, 6 & 14; SEQ ID NOs: 1, 8 & 10; SEQ ID NOs: 1, 8 & 12; SEQ IDNOs: 1, 8 & 14; SEQ ID NOs: 2, 8 & 10; SEQ ID NOs: 2, 8 & 12; SEQ IDNOs: 2, 8 & 14; SEQ ID NOs: 3, 8 & 10; SEQ ID NOs: 3, 8 & 12; SEQ IDNOs: 3, 8 & 14; SEQ ID NOs: 19, 22 & 28; SEQ ID NOs: 19, 22 & 30; SEQ IDNOs: 20, 22 & 28; SEQ ID NOs: 20, 22 & 30; SEQ ID NOs: 21, 22 & 28; SEQID NOs: 21, 22 & 30; SEQ ID NOs: 19, 24 & 28; SEQ ID NOs: 19, 24 & 30;SEQ ID NOs: 20, 24 & 28; SEQ ID NOs: 20, 24 & 30; SEQ ID NOs: 21, 24 &28; SEQ ID NOs: 21, 24 & 30; SEQ ID NOs: 19, 26 & 28; SEQ ID NOs: 19, 26& 30; SEQ ID NOs: 20, 26 & 28; SEQ ID NOs: 20, 26 & 30; SEQ ID NOs: 21,26 & 28; and, SEQ ID NOs: 21, 26 & 30. The methods and compositions mayalso comprise at least one capture probe oligomer that contains a targetbinding region consisting of SEQ ID NO: 35 or 36, optionally with a 3′tail sequence covalently attached to the target binding sequence.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the real-time fluorescent signal that was obtained fromamplification of varying copy levels of C. albicans 26S rRNA usingoligomer combination SEQ ID NOs: 1, 7, 11 & 17 as described in Example2.

FIG. 2 shows the real-time fluorescent signal that was obtained fromamplification of varying copy levels of C. albicans 26S rRNA usingoligomer combination SEQ ID NOs: 2, 7, 11 & 17 as described in Example2.

FIG. 3 shows the real-time fluorescent signal that was obtained fromamplification of varying copy levels of C. albicans 26S rRNA usingoligomer combination SEQ ID NOs: 19, 25, 29 & 33 as described in Example2.

DETAILED DESCRIPTION

Methods are disclosed for sensitively and specifically detecting thepresence of Candida albicans in an environmental, biopharmaceutical orbiological sample by detecting C. albicans nucleic acids. The methodsinclude performing a nucleic acid amplification of 26S rRNA sequencesand detecting the amplified product, typically by using a nucleic acidprobe that specifically hybridizes to the amplified product to provide asignal that indicates the presence of C. albicans in the sample. Theamplification step includes contacting the sample with one or moreamplification oligomers specific for a target sequence in 26S rRNA toproduce an amplified product if C. albicans rRNA is present in thesample. Amplification synthesizes additional copies of the targetsequence or its complement by using at least one nucleic acid polymeraseto extend the sequence from an amplification oligomer (a primer) using aC. albicans template strand. Preferred embodiments for detecting theamplified product use a hybridizing step that includes contacting theamplified product with at least one probe specific for an amplifiedsequence, e.g., a sequence contained in the target sequence that isflanked by a pair of amplification oligomers. The detecting step may beperformed after the amplification reaction is completed, or may beperformed simultaneous with the amplification reaction (sometimesreferred to as “real-time”). In preferred embodiments, the detectionstep detects the amplified product using a probe that is detected in ahomogeneous reaction, i.e., detection of the hybridized probe does notrequire removal of unhybridized probe from the mixture (e.g., U.S. Pat.Nos. 5,639,604 and 5,283,174, Arnold Jr. et al.). In preferredembodiments that detect the amplified product near or at the end of theamplification step, a linear probe hybridizes to the amplified productto provide a signal that indicates hybridization of the probe to theamplified sequence. In preferred embodiments that use real-timedetection, the probe is preferably a hairpin structure probe thatincludes a reporter moiety that provides the detected signal when theprobe binds to the amplified product. For example, a hairpin probe mayinclude a reporter moiety or label, such as a fluorophore (“F”),attached to one end of the probe and an interacting compound, such asquencher (“Q”), attached to the other end the hairpin structure toinhibit signal production when the hairpin structure is in the “closed”conformation and not hybridized to the amplified product, whereas adetectable signal results when the probe is hybridized to acomplementary sequence in the amplified product, thus converting theprobe to a “open” conformation. Examples of hairpin structure probeinclude a molecular beacon, molecular torch, or hybridization switchprobe and other forms (e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728,Lizardi et al.; U.S. Pat. Nos. 5,925,517 and 6,150,097, Tyagi et al.;U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274 and 6,361,945, Becker etal.; US Pub. No. 2006-0068417 A1, Becker et al.; and, US Pub. No.2006-0194240 A1, Arnold Jr. et al.).

To aid in understanding this disclosure, some terms used herein aredescribed below. Unless otherwise described, scientific and technicalterms used herein have the same meaning as commonly understood by thoseskilled in the relevant art based on technical literature, e.g., inDictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton etal., 1994, John Wiley & Sons, New York, N.Y.), The Harper CollinsDictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York,N.Y.), or Dorland's Illustrated Medical Dictionary, 30^(th) ed. (2003,W. B. Saunders, Elsevier Inc., Philadelphia, Pa.). Unless otherwisedescribed, techniques employed or contemplated herein are standardmethods well known in the art of molecular biology.

“Sample” includes any specimen that may contain Candida fungi orcomponents thereof, such as nucleic acids or nucleic acid fragments.Samples may be obtained from environmental sources, e.g., water, soil,slurries, debris, biofilms from containers of aqueous fluids, airborneparticles or aerosols, and the like, which may include processedsamples, such as those obtained from passing an environmental sampleover or through a filter, by centrifugation, or by adherence to amedium, matrix, or support. Samples may also be obtained from any stepalong a food supply chain to support food product safety or any stepalong a biopharmaceutical process stream to support sterile productdevelopment. “Biological samples” include any tissue or material derivedfrom a living or dead mammal, including humans, which may containCandida or target nucleic acid derived therefrom, e.g., respiratorytissue or exudates such as bronchoscopy, bronchoalveolar lavage (BAL) orlung biopsy, sputum, peripheral blood, plasma, serum, lymph node,gastrointestinal tissue, urine, exudates, or other body fluids. A samplemay be treated to physically or mechanically disrupt aggregates or cellsto release intracellular components, including nucleic acids, into asolution which may contain other components, such as enzymes, buffers,salts, detergents and the like.

“Nucleic acid” refers to a multimeric compound comprising nucleosides ornucleoside analogs which have nitrogenous heterocyclic bases, or baseanalogs, which are linked by phosphodiester bonds or other linkages toform a polynucleotide. Nucleic acids include RNA, DNA, or chimericDNA-RNA polymers, and analogs thereof. A nucleic acid “backbone” may bemade up of a variety of linkages, including one or more ofsugar-phosphodiester linkages, peptide-nucleic acid (PNA) bonds (PCT No.WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, orcombinations thereof. Sugar moieties of the nucleic acid may be eitherribose or deoxyribose, or similar compounds having known substitutions,e.g., 2′ methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine; The Biochemistry of the Nucleic Acids 5-36,Adams et al., ed., 11^(th) ed., 1992), derivatives of purine orpyrimidine bases, e.g., N⁴-methyl deoxyguanosine, deaza- or aza-purines,deaza- or aza-pyrimidines, pyrimidine bases having substituent groups atthe 5 or 6 position, purine bases having an altered or replacementsubstituent at the 2, 6 and/or 8 position, such as2-amino-6-methylaminopurine, O⁶-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO⁴-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or3-substituted pyrazolo[3,4-d]pyrimidine (U.S. Pat. Nos. 5,378,825,6,949,367 and PCT No. WO 93/13121). Nucleic acids may include “abasic”positions in which the backbone does not include a nitrogenous base forone or more residues (U.S. Pat. No. 5,585,481). A nucleic acid maycomprise only conventional sugars, bases, and linkages as found in RNAand DNA, or may include conventional components and substitutions (e.g.,conventional bases linked by a 2′ methoxy backbone, or a nucleic acidincluding a mixture of conventional bases and one or more base analogs).Nucleic acids also include “locked nucleic acids” (LNA), an analoguecontaining one or more LNA nucleotide monomers with a bicyclic furanoseunit locked in an RNA mimicking sugar conformation, which enhanceshybridization affinity toward complementary sequences in single-strandedRNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA)(Vester et al., 2004, Biochemistry 43(42): 13233-41). Methods forsynthesizing nucleic acids in vitro are well known in the art.

The interchangeable terms “oligomer” and “oligonucleotide” refer to anucleic acid having generally less than 1,000 nucleotides (nt),including polymers in a range having a lower limit of about 2 nt to 5 ntand an upper limit of about 500 nt to 900 nt. Preferred oligomers are ina size range having a lower limit of about 5 nt to 15 nt and an upperlimit of about 50 nt to 600 nt, and particularly preferred embodimentsare in a range having a lower limit of about 10 nt to 20 nt and an upperlimit of about 22 nt to 100 nt. Preferred oligomers are synthesized byusing any well known enzymatic or chemical method and purified bystandard methods, e.g., chromatography.

An “amplification oligomer” is an oligonucleotide that hybridizes to atarget nucleic acid, or its complement, and participates in a nucleicacid amplification reaction. An example of an amplification oligomer isa “primer” that hybridizes to a template nucleic acid and contains a 3′hydroxyl end that is extended by a polymerase in an amplificationprocess. Another example is an oligonucleotide that participates in orfacilitates amplification but is not extended by a polymerase, e.g.,because it has a 3′ blocked end. Preferred size ranges for amplificationoligomers include those that are about 10 to about 60 nt long andcontain at least about 10 contiguous bases, and more preferably at least12 contiguous bases that are complementary to a region of the targetnucleic acid sequence (or its complementary sequence). The contiguousbases are preferably at least 80%, more preferably at least 90%, andmost preferably about 100% complementary to the target sequence to whichthe amplification oligomer binds. An amplification oligomer mayoptionally include modified nucleotides or analogs, or optionally anadditional sequence that participates in an amplification reaction butis not complementary to or contained in the target or template sequence.For example, a “promoter-primer” is an oligonucleotide that includes a5′ promoter sequence that is non-complementary to the target nucleicacid but is adjacent or near to the target complementary sequence of theprimer. Those skilled in the art will understand that an amplificationoligomer that functions as a primer may be modified to include a 5′promoter sequence, and thus function as a promoter-primer, and apromoter-primer can function as a primer independent of its promotersequence, i.e., the oligonucleotide may be modified by removal of, orsynthesis without, its promoter sequence. An amplification oligomerreferred to as a “promoter-provider” includes a promoter sequence thatserves as a template for polymerization but the oligonucleotide is notextended from its 3′ end which is blocked and, therefore, not availablefor extension by polymerase activity.

“Amplification” refers to any known in vitro procedure for obtainingmultiple copies of a target nucleic acid sequence or fragments thereof,or its complementary sequence. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, e.g., by using anamplification oligonucleotide that hybridizes to and initiatespolymerization from an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, the polymerase chain reaction (PCR), ligase chainreaction (LCR), strand displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as Qβ-replicase (e.g., U.S. Pat. No. 4,786,600,Kramer et al.). PCR amplification uses a DNA polymerase, pairs ofprimers, and thermal cycling to synthesize multiple copies of twocomplementary strands of a dsDNA or from a cDNA (e.g., U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, Mullis et al.). LCR amplificationuses four or more different oligonucleotides to amplify a target and itscomplementary strand by using multiple cycles of hybridization,ligation, and denaturation (e.g., U.S. Pat. No. 5,427,930, Birkenmeyeret al.; U.S. Pat. No. 5,516,663, Backman et al.). SDA uses a primer thatcontains a recognition site for a restriction endonuclease and anendonuclease that nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, whereby amplification occurs in a seriesof primer extension and strand displacement steps (e.g., U.S. Pat. No.5,422,252, Walker et al.; U.S. Pat. No. 5,547,861, Nadeau et al.; U.S.Pat. No. 5,648,211, Fraiser et al.).

“Transcription-associated amplification” or “transcription-mediatedamplification” (TMA) refers to any type of nucleic acid amplificationthat uses an RNA polymerase to produce multiple RNA transcripts from anucleic acid template. These methods generally use an RNA polymerase, aDNA polymerase, nucleic acid substrates (dNTPs and rNTPs), and atemplate complementary oligonucleotide that includes a promotersequence, and optionally may include one or more other oligonucleotides.Variations of transcription-associated amplification are well known inthe art (e.g., disclosed in detail in U.S. Pat. Nos. 5,399,491 and5,554,516, Kacian et al.; U.S. Pat. No. 5,437,990, Burg et al.; PCT Nos.WO 88/01302 and WO 88/10315, Gingeras et al.; U.S. Pat. No. 5,130,238,Malek et al.; U.S. Pat. Nos. 4,868,105 and 5,124,246, Urdea et al.; PCTNo. WO 95/03430, Ryder et al.; and, US Pub. No. 2006-0046265 A1, Beckeret al.). TMA methods of Kacian et al. and a one primertranscription-associated method (US Pub. No. 2006-0046265 A1, Becker etal.) are preferred embodiments of transcription-associated amplificationmethods for use in detection of Candida target sequences as describedherein. Although preferred embodiments are illustrated by suchamplification reactions, a person of ordinary skill in the art willappreciated that amplification oligomers disclosed herein may be readilyused in other amplification methods that extend a sequence fromprimer(s) by using a polymerase.

“Probe” refers to a nucleic acid oligomer that hybridizes specificallyto a target sequence in a nucleic acid, preferably in an amplifiednucleic acid, under conditions that allow hybridization to permitdetection of the target sequence or amplified nucleic acid. Detectionmay either be direct (i.e., probe hybridized directly to its targetsequence) or indirect (i.e., probe linked to its target via anintermediate molecular structure). A probe's “target sequence” generallyrefers to a subsequence within a larger sequence (e.g., a subset of anamplified sequence) that hybridizes specifically to at least a portionof a probe by standard base pairing. A probe may include target-specificsequence and other sequences that contribute to the probe'sthree-dimensional conformation (e.g., described in U.S. Pat. Nos.5,118,801 and 5,312,728, Lizardi et al.; U.S. Pat. Nos. 6,849,412,6,835,542, 6,534,274 and 6,361,945, Becker et al.; and, US Pub. No.2006-0068417 A1 Becker et al.).

By “sufficiently complementary” is meant a contiguous sequence that iscapable of hybridizing to another sequence by hydrogen bonding between aseries of complementary bases, which may be complementary at eachposition in the sequence by standard base pairing (e.g., G:C, A:T or A:Upairing) or may contain one or more positions, including abasic ones,which are not complementary bases by standard hydrogen bonding.Contiguous bases are at least 80%, preferably at least 90%, and morepreferably about 100% complementary to a sequence to which an oligomeris intended to specifically hybridize. Sequences that are “sufficientlycomplementary” allow stable hybridization of a nucleic acid oligomer toits target sequence under the selected hybridization conditions, even ifthe sequences are not completely complementary. Appropriatehybridization conditions are well known in the art, can be predictedreadily based on base sequence composition, or can be determined byusing routine testing (e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989), §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57, particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57).

“Sample preparation” refers to any steps or methods that prepare asample for subsequent amplification and detection of Candida nucleicacids present in the sample. Sample preparation may include any knownmethod of concentrating components from a larger sample volume or from asubstantially aqueous mixture, e.g., by filtration or trapping ofairborne particles from an air sample or microbes from a water sample.Sample preparation may include lysis of cellular components and removalof debris, e.g., by filtration or centrifugation, and may include use ofnucleic acid oligomers to selectively capture the target nucleic acidfrom other sample components.

A “capture probe” or “capture oligomer” refers to at least one nucleicacid oligomer that joins a target sequence and an immobilized oligomerby using base pair hybridization to specifically or non-specificallycapture the target sequence. A preferred capture probe embodimentincludes two binding regions: a target sequence-binding region and animmobilized probe-binding region, usually on the same oligomer, althoughthe two regions may be present on different oligomers joined by one ormore linkers. For example, a first oligomer may include the immobilizedprobe-binding region and a second oligomer may include the targetsequence-binding region, and the two different oligomers are joined by alinker that joins the two sequences into a functional unit. Examples ofnon-specific target capture are described in U.S. application Ser. No.11/832,367, Becker et al.

An “immobilized probe” or “immobilized nucleic acid” refers to a nucleicacid that joins, directly or indirectly, a capture oligomer to animmobilized support. A preferred immobilized probe is an oligomer joinedto a support that facilitates separation of bound target sequence fromunbound material in a sample. Supports may include known materials, suchas matrices and particles free in solution, e.g., made up ofnitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane, polypropylene, metal and preferred embodiments are magneticallyattractable particles. Preferred supports are monodisperse magneticspheres (e.g., uniform size ±5%), to which an immobilized probe isjoined directly (via covalent linkage, chelation, or ionic interaction),or indirectly (via one or more linkers), where the linkage orinteraction between the probe and support is stable during hybridizationconditions.

“Separating” or “purifying” means that one or more components of amixture, such as a sample, are removed or separated from one or moreother components. Sample components include target nucleic acids in agenerally aqueous mixture (solution phase), which may include cellularfragments, proteins, carbohydrates, lipids, and other nucleic acids.Separating or purifying removes at least 70%, preferably at least 80%,and more preferably about 95% of the target nucleic acid from othermixture components.

A “label” refers to a molecular moiety or compound that is detected orleads to a detectable signal. A label may be joined directly orindirectly to a nucleic acid probe. Direct labeling can occur throughbonds or interactions that link the label to the probe, includingcovalent bonds or non-covalent interactions, e.g., hydrogen bonds,hydrophobic and ionic interactions, or formation of chelates orcoordination complexes. Indirect labeling can occur through use of abridging moiety or linker (e.g., antibody or additional oligomer), whichis either directly or indirectly labeled, and which may amplify thedetectable signal. Labels include any detectable moiety, such as aradionuclide, ligand (e.g., biotin, avidin), enzyme, enzyme substrate,reactive group, chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Preferredlabels include a “homogeneous detectable label” that provides adetectable signal in a homogeneous reaction in which bound labeled probein a mixture exhibits a detectable change that differs from that ofunbound labeled probe, e.g., stability or differential degradation(e.g., U.S. Pat. No. 5,283,174, Arnold et al.; U.S. Pat. No. 5,656,207,Woodhead et al.; U.S. Pat. No. 5,658,737, Nelson et al.). Preferredlabels include chemiluminescent compounds, preferably acridinium ester(“AE”) compounds that include standard AE and derivatives thereof(described in U.S. Pat. Nos. 5,656,207, 5,658,737 and 5,639,604).Methods of synthesis and attaching labels to nucleic acids and detectingsignals from labels are well known (e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989), Chpt. 10; U.S. Pat. Nos.5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333).

Methods are disclosed for amplifying and detecting Candida nucleic acid,specifically C. albicans 26S rRNA sequences or DNA encoding 26S rRNA.Disclosed are selected oligonucleotide sequences that specificallyrecognize target sequences of C. albicans 26S rRNA or theircomplementary sequences, or DNA encoding 26S rRNA. Such oligonucleotidesmay function as amplification oligomers, e.g., as primers,promoter-primers, blocked oligomers, and promoter-provider oligomers,whose functions are known (e.g., described in U.S. Pat. Nos. 4,683,195,4,683,202, 4,800,159, 5,399,491, 5,554,516 and 5,824,518; and, US Pub.No. 2006-0046265 A1). Other embodiments may function as probes to detectthe amplified C. albicans sequences.

Amplification methods that use transcription-mediated amplification(TMA) include the steps summarized herein (described in detail in U.S.Pat. Nos. 5,399,491, 5,554,516 and 5,824,518). The target nucleic acidthat contains the sequence to be amplified is provided assingle-stranded nucleic acid (e.g., ssRNA or ssDNA) or madesingle-stranded by conventional methods, e.g., temperature and/orchemical melting of double-stranded nucleic acid to provide asingle-stranded target nucleic acid. A promoter-primer bindsspecifically to its target sequence in the target nucleic acid and anenzyme with reverse transcriptase (RT) activity extends the 3′ end ofthe promoter-primer using the target strand as a template to make a cDNAof the target sequence, which is in an RNA:DNA duplex. Enzymatic RNaseactivity (e.g., RNaseH) digests the RNA strand of the RNA:DNA duplex anda second primer binds specifically to its target sequence on the cDNAstrand downstream from the promoter-primer end. The RT synthesizes a newDNA strand by extending the 3′ end of the second primer using the firstcDNA as a template to make a dsDNA that contains a functional promotersequence. An RNA polymerase specific for the promoter sequence theninitiates transcription to produce multiple RNA transcripts that are,e.g., about 100 to 1000 amplified copies (“amplicons”) of the initialtarget strand in the reaction. Amplification continues when the secondprimer binds specifically to its target sequence in each amplicon and RTmakes a DNA copy from the amplicon RNA template to produce an RNA:DNAduplex. RNase in the reaction digests the amplicon RNA from the RNA:DNAduplex and the promoter-primer binds specifically to its complementarysequence in the newly synthesized DNA. The RT extends the 3′ end of thepromoter-primer to create a dsDNA that contains a functional promoter towhich the RNA polymerase binds to transcribe additional amplicons thatare complementary to the initial target strand. These autocatalyticreactions make more amplicons repeatedly during the completeamplification reaction, resulting in about a billion-fold amplificationof the target sequence that was present in the sample. The amplifiedproducts may be detected during amplification, i.e., in real-time, or atcompletion of the amplification reaction by using a probe that bindsspecifically to a target sequence in the amplified products. Signaldetected from the bound probes indicates the presence of the targetnucleic acid in the sample.

Another transcription-associated amplification method summarized hereinuses one primer and one or more additional amplification oligomers toamplify nucleic acids in vitro by making transcripts (amplicons) thatindicate the presence of the target nucleic acid in a sample (describedin detail in US Pub. No. 2006-0046265 A1, Becker et al.). Briefly, thissingle-primer method uses a primer or “priming oligomer”, a“promoter-provider” oligomer that is modified to prevent syntheticextension from its 3′ end (typically, by including a 3′-blocking moiety)and, optionally, a binding molecule (e.g., a 3′-blocked extenderoligomer) to terminate elongation of a cDNA from the target strand. Thismethod includes the steps of binding the target RNA that contains thetarget sequence with a primer and, optionally, a binding molecule. Theprimer hybridizes to the 3′ end of the target strand and enzymatic RTactivity initiates primer extension from the 3′ end of the primer toproduce a cDNA, to make a duplex of the new strand and the target strand(RNA:cDNA duplex). When a binding molecule is included in the reaction,such as a 3′-blocked oligomer, it binds to the target strand next to the6′ end of the target sequence to be amplified. When the primer isextended by DNA polymerase activity of RT to produce the cDNA strand,polymerization stops when the primer extension product reaches thebinding molecule on the target strand and, thus, the 3′ end of the cDNAis determined by the position of the binding molecule on the targetstrand, making the 3′ end of the cDNA complementary to the 5′ end of thetarget sequence. The RNA:cDNA duplex is separated, e.g., by RNase Hdegradation of the RNA strand, or by using conventional strandseparation methods. Then, the promoter-provider oligomer hybridizes tothe cDNA strand near its 3′ end. The promoter-provider oligomer includesa 5′ promoter sequence, a 3′ region complementary to a sequence in the3′ region of the cDNA, and a modified 3′ end that includes a blockingmoiety to prevent initiation of DNA synthesis from the 3′ end of thepromoter-provider oligomer. In the duplex made of the promoter-provideroligomer and the cDNA strand, the 3′-end of the cDNA is extended by DNApolymerase activity of the RT enzyme, using the promoter oligomer as atemplate to add a promoter sequence to the cDNA, to make a functionaldouble-stranded promoter. An RNA polymerase specific for the functionalpromoter sequence then binds to the promoter and transcribes RNAtranscripts complementary to the cDNA which are substantially identicalto the target region sequence that was amplified from the initial targetstrand. The amplified RNA transcripts then serve as substrates in theamplification process by binding the primer and serving as a templatefor further cDNA production. This method ultimately produces manyamplicons from the initial target nucleic acid present in the sample,i.e., it makes multiple copies of the target sequence. In embodiments ofthe method that do not include the binding molecule, the cDNA made fromthe primer has an indeterminate 3′ end, but the other steps proceed asdescribed above.

Detection of the amplified products may be accomplished by a variety ofmethods. The amplified nucleic acids may be associated with a surface toproduce a detectable physical change, such as an electrical signal.Amplified nucleic acids may be concentrated in or on a matrix anddetected by detecting a signal from the concentrated nucleic acid or anassociated dye (e.g., an intercalating agent such as ethidium bromide orcyber green). Nucleic acids in solution may be detected by detecting anincreased dye association in the solution phase. Preferred embodimentsdetect nucleic acid probes that are complementary to a sequence in theamplified product and form a probe:amplified product complex thatprovides a detectable signal (e.g., U.S. Pat. Nos. 5,424,413 and5,451,503, Hogan et al.; and, U.S. Pat. No. 5,849,481, Urdea et al.).Directly or indirectly labeled probes that specifically associate withthe amplified product provide a detectable signal to indicate thepresence of the target nucleic acid in the sample. For example, if asample contains a target nucleic acid that is Candida albicans 26S rRNA,the amplified product contains the target sequence in or a complementarysequence of the C. albicans 26S rRNA, and the probe binds directly orindirectly to the amplified product's target sequence to produce asignal that indicates the presence of C. albicans in the sample.

Preferred probe embodiments that hybridize specifically to the amplifiedproduct sequences may be oligomers of DNA, RNA, or a mixture of DNA andRNA nucleotides, which may be synthesized with a modified backbone,e.g., a synthetic oligonucleotide that includes one or more 2′-methoxysubstituted RNA groups. Probes for detection of amplified Candida rRNAsequences may be unlabeled and detected indirectly (e.g., by binding toanother binding partner that is detected) or may be labeled with a labelthat results in a detectable signal. Preferred embodiments include labelcompounds that emit a detectable light signal, e.g., fluorophores orluminescent compounds detected in a homogeneous mixture. A probe mayinclude more than one label and/or more than one type of label, ordetection may rely on using a mixture of probes in which each probe islabeled with a compound that produces a detectable signal (e.g., U.S.Pat. Nos. 6,180,340 and 6,350,579). Labels may be attached to a probe byany of a variety of known means, e.g., covalent linkages, chelation, andionic interactions, but preferred embodiments covalently link the labelto the oligonucleotide. Probes may be substantially linearoligonucleotides, i.e., lacking conformations held by intramolecularbonds, or may be include functional conformational structures, i.e.,conformations such those found in hairpin structure probes held togetherby intramolecular hybridization. Preferred embodiments of linearoligomers generally include a chemiluminescent label, preferably an AEcompound.

Hairpin probes are preferably labeled with any of a variety of differenttypes of interacting labels, in which one interacting member is usuallyattached to the 5′ end of the probe and the other interacting member isattached to the 3′ end of the probe. Such interacting members includethose often referred to as a reporter dye/quencher pair, aluminescent/quencher pair, luminescent/adduct pair, Forrester energytransfer pair, or dye dimer. A luminescent/quencher pair may be made upof one or more luminescent labels, such as chemiluminescent orfluorescent labels, and one or more quenchers. In preferred embodiments,a hairpin probe is labeled at one end with a fluorophore (“F”) thatabsorbs light at a particular first wavelength or range and emits lightat a second emission wavelength or range and labeled at the other endwith a quencher (“Q”) that dampens, partially or completely, signalemitted from the excited F when Q is in proximity with the fluorophore.Such a hairpin probe may be referred to as labeled with afluorescent/quencher (F/Q) pair. Fluorophores are well known compoundsthat include, e.g., acridine, fluorescein, sulforhodamine 101,rhodamine, 5-(2′-aminoethyl)aminoaphthaline-1-sulfonic acid (EDANS),Texas Red, Eosine, Bodipy and lucifer yellow (Tyagi et al., NatureBiotechnology 16:49-53, 1998). Quenchers are well known and include,e.g., 4-(4′-dimethyl-amino-phenylaxo)benzoic acid (DABCYL), thallium,cesium, and p-xylene-bis-pyridinium bromide. Different F/Q combinationsare known and many combinations may function together, e.g., DABCYL withfluorescein, rhodamine, or EDANS. Other combinations of labels forhairpin probes include a reporter dye, e.g., FAM™, TET™, JOE™, VIC™combined with a quencher such as TAMRA™ or a non-fluorescent quencher. Afunctional F/Q combination may be determined by using routine testingusing known procedures.

A preferred embodiment of a hairpin probe is a “molecular torch” thatdetects an amplified product to indicate the presence of a targetCandida sequence in a sample after the amplification step. A moleculartorch includes: (1) a target detection means that hybridizes to thetarget sequence, resulting in an open conformation; (2) a torch closingmeans that hybridizes to the target detecting means in the absence ofthe target sequence, resulting in a closed conformation; and (3) ajoining means that joins the target detection means and the torchclosing means (described in detail in U.S. Pat. Nos. 6,849,412,6,835,542, 6,534,274 and 6,361,945). A torch probe in open conformationresults in a detectable signal that indicates the presence of theamplified target sequence, whereas the closed conformation produces anamount of signal that is distinguishable from that of the openconformation indicating that the target sequence is not present. Anotherpreferred hairpin probe embodiment is a “molecular beacon” that includesa label on one arm of the hairpin sequence, a quencher on the other arm,and a loop region joining the two arms (described in detail in U.S. Pat.Nos. 5,118,801 and 5,312,728). Methods for using such hairpin probes arewell known in the art.

Oligomers that are not extended by a nucleic acid polymerase include ablocker group that replaces the 3′ OH to prevent enzyme-mediatedextension of the oligomer in an amplification reaction. Blockedamplification oligomers and/or blocked detection probes present duringamplification (for real-time detection) preferably lack a 3′ OH butinclude one or more blocking groups located at or near the 3′ end. Ablocking group is covalently attached to the 3′ terminus of theoligonucleotide or is located near the 3′ end, preferably within fiveresidues of the 3′ end, and is sufficiently large to limit binding of apolymerase to the oligomer. Many different chemical groups may be usedas a blocking moiety, e.g., alkyl groups, non-nucleotide linkers,alkane-diol dideoxynucleotides, and cordycepin.

A preferred method for detection of Candida albicans sequences uses atranscription-associated amplification with a hairpin probe, e.g.molecular torch or molecular beacon, because the probe may be addedbefore amplification, and detection is carried out without furtheraddition of reagents. For example, a probe may be designed so that theT_(m) of the hybridized arms of the hairpin probe (e.g., target bindingdomain:target closing domain complex of a molecular torch) is higherthan the amplification reaction temperature to prevent the probe fromprematurely binding to amplified target sequences. After an interval ofamplification, the mixture is heated to open the torch probe arms andallow the target binding domain to hybridize to its target sequence inthe amplified product. The solution is then cooled to close probes notbound to amplified products, which closes the label/quencher (F/Q) pair,allowing detection of signals from probes hybridized to the amplifiedtarget sequences in a homogeneous reaction. For example, the mixturecontaining the F/Q labeled hairpin probe is irradiated with theappropriate excitation light and the emission signal is measured.

In other embodiments, the hairpin detection probe is designed so thatamplified products preferentially hybridize to the target binding domainof the probe during amplification, thereby changing the hairpin from itsclosed to open conformation as amplification progresses. Theamplification reaction mixture is irradiated at intervals during theamplification reaction to detect the emitted signal from the open probesduring amplification, i.e., in real-time.

Preparation of samples for amplification of Candida sequences mayinclude separating and/or concentrating organisms contained in a samplefrom other sample components, e.g., filtration of particulate matterfrom air, water or other types of samples. Sample preparation may alsoinclude chemical, mechanical, and/or enzymatic disruption of cells torelease intracellular contents, including Candida 26S rRNA or DNAencoding the 26S rRNA. Sample preparation may include a step of targetcapture to specifically or non-specifically separate the target nucleicacids from other sample components. Non-specific target preparationmethods may selectively precipitate nucleic acids from a substantiallyaqueous mixture, adhere nucleic acids to a support that is washed toremove other sample components, or use other means to physicallyseparate nucleic acids, including Candida nucleic acid, from a mixturethat contains other components. Other non-specific target preparationmethods may selectively separate RNA, including Candida 26S rRNA, fromDNA in a sample.

In a preferred embodiment, Candida 26S rRNA or DNA encoding 26S rRNA areselectively separated from other sample components by specificallyhybridizing the Candida nucleic acid to a capture oligomer specific forthe Candida target sequence to form a target sequence:capture probecomplex that is separated from sample components. A preferred embodimentof specific target capture binds the Candida target:capture probecomplex to an immobilized probe to form a target:captureprobe:immobilized probe complex that is separated from the sample and,optionally, washed to remove non-target sample components. The captureprobe includes a sequence that specifically binds to the Candida targetsequence in 26S rRNA or in DNA encoding 26S rRNA and also includes aspecific binding partner that attaches the capture probe with its boundtarget sequence to a support (e.g., matrix or particle), whichfacilitates separating the target sequence from the sample components.In a preferred embodiment, the specific binding partner of the captureprobe is a 3′ tail sequence that is not complementary to the Candidatarget sequence but that hybridizes to a complementary sequence on animmobilized probe attached to the support. Preferred 3′ tail sequencesare substantially homopolymeric 10 to 40 nt sequences (e.g., A₁₀ to A₄₀)that bind to a complementary immobilized sequence (e.g., poly-T)attached to the support. Target capture occurs in a solution phasemixture that contains capture oligomers that hybridize specifically tothe Candida target nucleic acid under hybridizing conditions, usually ata temperature higher than the T_(m) of the tail sequence:immobilizedprobe sequence duplex. The Candida target:capture probe complex iscaptured by adjusting the hybridization conditions so that the captureprobe tail then hybridizes to the immobilized probe, and the entirecomplex on the support is separated from the other sample components.The support with the attached complex that includes the Candida targetsequence may be washed to further remove other sample components.Preferred supports are particulate, such as paramagnetic beads, so thatparticles with the complex that includes the captured Candida targetsequence may be suspended in a washing solution and retrieved from thewashing solution by using magnetic attraction. In other embodiments, thecapture probe may bind nonspecifically to nucleic acids in the sample,including the Candida target sequence, and then similar steps ofattaching the capture probe:nucleic acid complexes to a support andseparating the captured complexes on the support are performed. Whethertarget capture is specific or non-specific for the Candida targetsequence, the captured nucleic acids are then subjected to in vitroamplification specific for the intended Candida target sequence. Tolimit the number of handling steps, Candida target nucleic acid may beamplified by mixing the Candida target sequence in the captured complexon the support with amplification reagents, or a primer may be includedin the target capture reaction mixture, thus allowing the Candidaspecific primer and target sequences to hybridize during target captureand be separated together from the sample in the captured complex.

Assays for detection of Candida nucleic acid may optionally include anon-Candida internal control (IC) nucleic acid that is amplified anddetected in the same assay reaction mixtures by using amplification anddetection oligomers specific for the IC sequence. Amplification anddetection of a signal from the amplified IC sequence demonstrates thatthe assay reagents, conditions, and procedural steps were properly usedand performed in the assay if no signal is obtained for the intendedtarget Candida nucleic acid (e.g., samples that provide negative resultsfor C. albicans). The IC may be used as an internal calibrator for theassay when a quantitative result is desired, i.e., the signal obtainedfrom the IC amplification and detection is used to set a parameter usedin an algorithm for quantitating the amount of Candida nucleic acid in asample based on the signal obtained for amplified an Candida targetsequence. A preferred IC embodiment is a randomized sequence that hasbeen derived from a naturally occurring source (e.g., an HIV sequencethat has been rearranged in a random manner). A preferred IC may be anRNA transcript isolated from a naturally occurring source or synthesizedin vitro, such as by making transcripts from a cloned randomizedsequence such that the number of copies of IC included in an assay maybe accurately determined. The primers and probe for the IC targetsequence are designed and synthesized by using any well known methodprovided that the primers and probe function for amplification of the ICtarget sequence and detection of the amplified IC sequence usingsubstantially the same assay conditions used to amplify and detect theCandida target sequence and the IC components in the assay do notinterfere with those used to amplify and detect the Candida targetsequence. In preferred embodiments that include a target capture-basedpurification step, a target capture probe specific for the IC target isincluded in the target capture step so that the IC is treated in thesame conditions as used for the intended Candida analyte in all of theassay steps.

EXAMPLES

For amplification and detection of target sequences in 26S rRNAsequences (which include 26S rRNA and DNA encoding 26S rRNA) of Candidaalbicans, oligomers were designed that act as amplification oligomersand detection probes by comparing known sequences of 26S rRNA or genesequences encoding 26S rRNA and selecting sequences that are common toC. albicans isolates, but preferably are not completely identical to 26SrRNA sequences of other Candida species or other eukaryotic organisms.Sequence comparisons were conducted by using known 26S rRNA sequences(rRNA or genes) of Candida species (C. dubliniensis, C. tropicalis andC. glabrata) and of other fungal species (S. cerevislae, S. barnettii,S. exiguus, S. spencerorum, K. lodderae, S. rosinii, S. unisporus, S.servezzii, K. africanus, S. dairenensis, S. castellii, S. paradoxus, S.bayanus, K. yarrowil, K. polysporus, E. gossypii and E. fibuliger).Specific oligonucleotide sequences were selected, synthesized in vitroand characterized with purified rRNA from fungi using standardlaboratory methods. The selected oligomers were further tested by usingdifferent combinations of the amplification oligomers in amplificationreactions with whole cell lysates or total RNA purified from fungi grownin culture, to determine the relative efficiencies of amplification ofthe target sequences by using the selected amplification oligomers. Theefficiencies of different combinations of oligomers were monitored bydetecting the amplified products of the amplification reactions;generally by binding a labeled probe oligomer to the amplified productsand detecting a signal that indicated the presence of amplified product.

Preferred embodiments of the selected amplification oligomers for C.albicans 26S rRNA target sequences are shown in Table 1. Amplificationoligomers include those that may function as primers, promoter-primers,and/or promoter-provider oligomers. For the latter two, promotersequences are shown in lower case in Table 1. Some oligomer embodimentsinclude only the target-specific sequence of a correspondingpromoter-primer or promoter-provider oligomer, e.g., SEQ ID NO: 4 is atarget-specific sequence that is identical to the target-specificsequence contained in SEQ ID NO: 5, which includes a 5′ promotersequence. Those skilled in the art will appreciate that thetarget-specific sequences listed in Table 1 may optionally be attachedto the 3′ end of any known promoter sequence to function as apromoter-primer or promoter-provider with the appropriate RNA polymerasefor the chosen promoter sequence. An example of a promoter sequencespecific for the RNA polymerase of bacteriophage T7 is SEQ ID NO: 37(AATTTAATACGACTCACTATAGGGAGA). Preferred embodiments of amplificationoligomers may include a mixture of DNA and RNA bases, and 2′ methoxy RNAgroups, e.g., oligomers of SEQ ID NOs: 1-3 and 19-21 may include RNAbases and 2′ methoxy linkages at the first four positions from the 5′end. Embodiments of amplification oligomers may be modified bysynthesizing the oligomer with the 3′ end blocked to make the oligomeroptimal for functioning as a blocking molecule or promoter-provideroligomer in a single-primer transcription-associated amplificationreaction. Preferred embodiments of 3′-blocked oligomers include those ofSEQ ID NOs: 5, 7, 9, 16-18, 23, 25, 27 and 32-34 that include a blockedC near or at the 3′ end.

TABLE 1 SEQ ID Sequence NO. CAGATTCCCCTTGTCCGTACC 1GACAGTCAGATTCCCCTTGTCC 2 CACUTTCTGACCATCACAATGC 3GATAAGGATTGGCTCTAAGGATCGGGTGTC 4aatttaatacgactcactatagggagaGATAAGGATTGGCTCT 5 AAGGATCGGGTGTCGCGGTGACTGTTGGCGGGCTGTTTC 6 aatttaatacgactcactatagggagaGCGGTGACTGTTGGCG7 GGCTGTTTG GCTGTTTCACGACGGACTGCTGGTGGATG 8aatttaatacgactcactatagggagaGCTGTTTCACGACGGA 9 CTGCTGGTGGATGCUUAUCCCGAAGUUACGGAUC 16 CACCGCCGCGUCUACACAAG 17 GAAACAGCCCGCCAACAGUCAC18 CCUGCGTTATCGTTTAACAGATGTGCC 19 CAUGAGTCCCCCTTAGGACACCTGC 20GGAGATTTCTGTTCTCCATGAGTCC 21 GGAGGGTGTAGAATAAGTGGGAGCTTCG 22aatttaatacgactcactatagggagaGGAGGGTGTAGAATAA 23 GTGGGAGCTTCGGCTTCGGCGCCGGTGAAATACCACTACC 24aatttaatacgactcactatagggagaGCTTCGGCGCCGGTGA 25 AATACCACTACCCTTATTCAATGAAGCGGAGCTGGAGGTC 26aatttaatacgactcactatagggagaCTTATTCAATGAAGCG 27 GAGCTGGAGGTCCCUCCAUGUCUUUUCACAAUG 32 GAAGCUCCCACUUAUUCUAC 33 GAAUAAGUAAAAAAACUAUAG34

Preferred embodiments of the selected detection probe oligomers fordetecting amplified products of 26S rRNA sequences or DNA encoding 26SrRNA are shown in Table 2. Preferred detection probe embodiments areoligomers that form hairpin configurations by intramolecularhybridization of the probe sequence, of which preferred embodiments arethose of SEQ ID NOs: 11, 13, 15, 29 and 31. Preferred hairpin probeoligomers are synthesized with a fluorescent label attached at one endand a quencher compound attached at the other end of the sequence.Embodiments of hairpin probes may be labeled with a 5′ fluorophore and a3′ quencher, e.g., 5′ fluoroscein label with 3′ DABCYL quencher. Someembodiments of hairpin oligomers include a non-nucleotide linker moietyat selected positions within the sequence, e.g., oligomers that includean abasic 9-carbon (“C9”) linker located in: SEQ ID NO: 11 between nt 5and nt 6, SEQ ID NO: 13 between nt 20 and nt 21, SEQ ID NO: 15 betweennt 17 and nt 18, SEQ ID NO: 29 between nt 16 and nt 17, and SEQ ID NO:31 between nt 20 and nt 21.

TABLE 2 Sequence SEQ ID NO. CCAUAAAGACCUACCAAGCGUG 10cacgcCCAUAAAGACCUACCAAGCGUG 11 CCGGACGGCGAUAAAGACCU 12CCGGACGGCGAUAAAGACCUuccgg 13 CGGACGGCCAUAAAGAC 14 CGGACGGCCAUAAAGACguccg15 CCCAGAGGGCUUAAUG 28 CCCAGAGGGCUUAAUGcuggg 29 CGGAUCGCCCAGAGGGCUUA 30CGGAUCGCCCAGAGGGCUUAauccg 31

Embodiments of non-specific and specific capture probe oligomers for usein sample preparation to separate Candida 26S rRNA target nucleic acidsfrom other sample components include those that contain the sequences ofSEQ ID NO: 35 (kkkkkkkkkkkkkkkkkk) and SEQ ID NO: 36(CGAGGCAUUUGGCUACCUUAAGAG), respectively. Preferred embodiments of thecapture probes include a 3′ tail region covalently attached to thesequence to serve as a binding partner that binds a hybridizationcomplex made up of the target nucleic acid and the capture probe to animmobilized probe on a support. Preferred embodiments of capture probesthat include the sequences of SEQ ID NOs: 35 and 36 further include 3′tail regions made up of substantially homopolymeric sequences, e.g., adT₃A₃₀ sequence.

Reagents used in target capture and amplification described in theexamples herein generally include one or more of the following. LysisReagent: 20 mM Lithium Succinate, 0.1% (w/v) LLS, and 1 mM LiOH. TargetCapture Reagent: 250 mM HEPES, 310 mM LiOH, 1.88 M LiCl, 100 mM EDTA, pH6.4, and 250 μg/ml of paramagnetic particles (0.7-1.05 μparticles,SERA-MAG™ MG-CM, Seradyn, Inc., Indianapolis, Ind.) with covalentlybound (dT)₁₄ oligomers. Wash Solution: 10 mM HEPES, 150 mM NaCl, 6.5 mMNaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methyl paraben, 0.01%(w/v) propyl paraben, and 0.1% (w/v) sodium lauryl sulfate, pH 7.5.Amplification reagent: 46.1 mM HEPES, 86.2 mM Trehalose Dihydrate, 33 mMKCl, 30.6 mM MgCl₂, 1.7 mM NaOH, 0.5 mM of each dNTP (dATP, dCTP, dGTP,dTTP), 10 mM rATP, 2 mM rCTP, 2 mM UTP, 12 mM rGTP, 0.4% ethanol, 0.1%methylparaben, 0.02% propylparaben. Enzyme Reagent: 58 mM HEPES, 3.03%(w/v) Trehalose Dihydrate, 50 mM N-acetyl-L-cysteine, 10% (v/v) TritonX-100, 1.04 mM EDTA Disodium Dihydrate, 20% Glycerol, 120 mM KCl, andabout 360 RTU/μl of MMLV reverse transcriptase (MMLV-RT) and about 80U/μl of T7 RNA polymerase. One reverse transcriptase unit (“RTU”) ofactivity for MMLV reverse transcriptase is defined as the incorporationof 1 nmol dTMP into DE81 filter-bound product in 20 minutes at 37° C.using (poly(rA)-p(dT)₁₂₋₁₈) as the substrate, and for T7 RNA polymerase,one unit (“U”) of activity is defined as the production of 5.0 fmol RNAtranscript in 20 minutes at 37° C. All of the reagent addition andmixing steps may be performed manually, or by using a combination ofmanual and automated steps, or by using a completely automated system.Amplification methods that use single-primer transcription-associatedamplification use procedures substantially as disclosed in US Pub. No.2006-0046265 A1, Becker et al. Methods for using hairpin probes havebeen disclosed in detail in U.S. Pat. Nos. 6,849,412, 6,835,542,6,534,274 and 6,361,945.

Different amplification oligomer combinations were made from thoselisted in Table 1 and were tested in single-primertranscription-associated amplifications as described above, using wholecell lysates or ribosomal RNA isolated from C. albicans and other fungias target nucleic acid. Amplified products were detected by usinghairpin probes (molecular torches) from those listed in Table 2 labeledwith a fluorophore (5′ fluorescein) and 3′ quencher (DABCYL), detectingthe fluorescence emitted when the probe bound to amplified sequences.

Example 1 Amplification and Detection Probe Oligomer Combinations

Amplification and detection of a C. albicans 26S rRNA target sequencewas demonstrated in real-time by using a probe that hybridizes to theamplified product during the amplification reaction. Amplification wasperformed by using a single-primer transcription-associatedamplification procedure substantially as described in detail in US Pub.No. 2006-0046265 A1, conducted by using some of the selectedamplification oligomers. Each of the assays was performed in anamplification reaction (0.040 ml total volume) that contained the C.albicans target RNA and amplification reagents substantially asdescribed for TMA reactions but with a promoter-provider oligomer (12pmol per reaction), a primer oligomer (6 pmol per reaction), a blockeroligomer (0.5 pmol per reaction), and a hairpin probe (molecular torchat 6 pmol per reaction). Reaction mixtures containing the amplificationoligomers, target and amplification reagents (but not enzymes) werecovered to prevent evaporation, incubated 10 min at 60° C., then 5 minat 42° C., then enzymes were added (10 μl vol) and the reactions weremixed and incubated for 50 min at 42° C., measuring fluorescence every20 sec during the amplification reaction after enzyme addition.

TABLE 3 Oligomer Average Measured Time-of-Emergence (min) Combinations 0SEQ ID NOs copies/rxn 3 × 10⁴ copies/rxn 3 × 10⁷ copies/rxn 1, 5, 11, 16ND ND 18.9 1, 5, 13, 16 ND ND ND 1, 5, 15, 16 ND ND 18.6 2, 5, 11, 16 NDND 20.2 2, 5, 13, 16 ND ND 19.8 2, 5, 15, 16 ND ND 19.3 3, 5, 11, 16 NDND ND 3, 5, 13, 16 ND ND ND 3, 5, 15, 16 ND ND ND 1, 7, 11, 17 ND 21.914.5 1, 7, 13, 17 ND 23.8 18.2 1, 7, 15, 17 ND 22.5 15.7 2, 7, 11, 17 ND23.8 14.7 2, 7, 13, 17 ND 24.4 18.1 2, 7, 15, 17 ND 21.7 15.5 3, 7, 11,17 ND ND ND 3, 7, 13, 17 ND ND ND 3, 7, 15, 17 ND ND ND 1, 9, 11, 18 ND19.2 12.3 1, 9, 13, 18 ND 20.0 15.7 1, 9, 15, 18 ND 18.3 13.1 2, 9, 11,18 ND ND 13.2 2, 9, 13, 18 ND ND 15.9 2, 9, 15, 18 ND ND 13.6 3, 9, 11,18 ND ND ND 3, 9, 13, 18 ND ND ND 3, 9, 15, 18 ND ND ND 19, 23, 29, 32ND ND ND 19, 23, 31, 32 ND ND ND 20, 23, 29, 32 ND ND ND 20, 23, 31, 32ND ND ND 21, 23, 29, 32 ND ND ND 21, 23, 31, 32 ND ND ND 19, 25, 29, 33ND 19.7 15.0 19, 25, 31, 33 ND 24.8 21.1 20, 25, 29, 33 ND ND 27.6 20,25, 31, 33 ND ND 30.0 21, 25, 29, 33 ND ND 33.1 21, 25, 31, 33 ND ND ND19, 27, 29, 34 ND 30.6 23.4 19, 27, 31, 34 ND 30.9 27.5 20, 27, 29, 34ND ND ND 20, 27, 31, 34 ND ND ND 21, 27, 29, 34 ND ND ND 21, 27, 31, 34ND ND ND ND denotes not detectedOligomer combinations SEQ ID NOs: 1, 7, 11 & 17, SEQ ID NOs: 2, 7, 11&17 and SEQ ID NOs: 19, 25, 29 & 33 showed the best sigmoidal curves atboth 3×10⁴ copies and 3×10⁷ copies and clear distinction on emergencetime between the two levels. No amplification was detected on any of theblanks (0 copies).

Example 2 Sensitivity

The preferred oligomer combinations from Example 1 were subsequentlytested for C. albicans 26S rRNA target sensitivity.

TABLE 4 Average Measured Time-of-Emergence (min) Oligomer OligomerOligomer Combination Combination Combination Target SEQ ID NOs: SEQ IDNOs: SEQ ID NOs: (copies/rxn) 1, 7, 11, 17 2, 7, 11, 17 19, 25, 29, 33 0ND ND ND 3 ND ND ND 3 × 10 ND ND ND 3 × 10² 17.8 23.4 28.7 3 × 10³ 21.821.6 25.5 3 × 10⁴ 23.1 23.6 22.7 3 × 10⁵ 20.0 20.5 19.7 3 × 10⁶ 17.918.0 17.2 ND denotes not detectedReferring to Table 4 in conjunction with FIGS. 1-3, all three oligomercombinations unequivocally detected down to at least 3×10⁴ copies(approximately 3 cells). Oligomer combination SEQ ID NOs: 19, 25, 29 &33 unequivocally detected down to 3×10³ copies (approximately 0.3 cell).

Example 3 Specificity

Oligomer combinations SEQ ID NOs: 1, 7, 11 & 17 and SEQ ID NOs: 19, 25,29 & 33 were also tested for C. albicans 26S rRNA target specificity.

TABLE 5 Oligomer Combination Oligomer Combination SEQ ID NOs: SEQ IDNOs: 1, 7, 11, 17 19, 25, 29, 33 Average Average Measured MeasuredTime-of- Target Time-of- Target Emergence (copies/ Emergence Organism(copies/rxn) (min) rxn) (min) C. albicans 3 × 10⁹ 15.4*/10.4** 3 × 10⁹15.5*/9.9** C. guillermondii 3 × 10⁹ ND* 3 × 10⁹ ND* C. kruseii 3 × 10⁹ND* 3 × 10⁹ ND* C. parapisilosis 3 × 10⁹ 15.2* 3 × 10⁹ ND* C. tropicalis3 × 10⁹ 15.0* 3 × 10⁹ ND* C. glabrata 3 × 10⁹ 14.6** 3 × 10⁹ ND** C.kefur 3 × 10⁹ 14.0** 3 × 10⁹ ND** ND denotes not detected *denotesresult from 1^(st) experiment **denotes result from 2^(nd) experimentOligomer combination SEQ ID NOs: 19, 25, 29 & 33 demonstratedspecificity for C. albicans without detection of C. guillermondii, C.kruseil, C. parapisilosis, C. tropicalis, C. glabrata and C. kefur.

Example 4 Target Capture

Non-specific and specific target capture of C. albicans 26S rRNA werecompared using oligomer combinations SEQ ID NOs: 1, 7, 11 & 17 and SEQID NOs: 19, 25, 29 & 33 for amplification and detection.

TABLE 6 Average Measured Time-of-Emergence (min) Oligomer CombinationOligomer Combination SEQ ID NOs: SEQ ID NOs: 1, 7, 11, 17 19, 25, 29, 33TC Oligomer TC Oligomer TC Oligomer TC Oligomer Target SEQ ID SEQ ID SEQID SEQ ID (copies/rxn) NO: 35 NO: 36 NO: 35 NO: 36 0  ND 17.7 ND ND 10²ND  7.1 25.8  4.8 10³ 11.2 ND 21.1 19.6 10⁴ 11.7 20.8 18.5 17.9 10⁵ 18.017.7 15.7 16.1 10⁶ 16.5 15.9 14.2 14.1 10⁷ 14.7 14.5 12.5 12.5 10⁸ 12.912.9 11.0 10.9 ND denotes not detectedNon-specific and specific target capture demonstrated comparableperformance. All oligomer combinations unequivocally detected down to10⁴ copies (approximately 1 cell). Oligomer combination SEQ ID NOs: 19,25, 29 & 33 unequivocally detected down to 10³ copies (approximately 0.1cell).

1. A method of detecting Candida albicans in a sample, comprising thesteps of: mixing a sample that contains a C. albicans target nucleicacid that is a 26S rRNA sequence or DNA encoding the 26S rRNA sequencewith a first amplification oligomer of up to 100 nucleotides in lengthcomprising a target binding region consisting of SEQ ID NO: 19 and asecond amplification oligomer of up to 100 nucleotides in lengthcomprising a target binding region consisting of SEQ ID NO: 24;providing an enzyme with nucleic acid polymerase activity and nucleicacid precursors to make an amplification mixture that includes the firstand second amplification oligomers and the C. albicans target nucleicacid; elongating in vitro a 3′ end of at least one of the amplificationoligomers hybridized to the C. albicans target nucleic acid by using theenzyme with nucleic acid polymerase activity and the C. albicans targetnucleic acid as a template to produce an amplified product; anddetecting the amplified product by hybridizing the amplified productspecifically to a detection probe oligomer of up to 100 nucleotides inlength comprising a target binding sequence consisting of SEQ ID NO: 28to indicate the presence Candida albicans in the sample.
 2. The methodof claim 1, further comprising a sample processing step that capturesthe C. albicans target nucleic acid from the sample before the mixingstep.
 3. The method of claim 2, wherein the sample processing step usesa capture probe oligomer that contains a target binding sequenceconsisting of SEQ ID NO: 35 or 36, wherein the target binding sequenceis optionally covalently attached to a 3′ tail sequence.
 4. Acomposition for detecting Candida albicans 26S rRNA sequence or DNAencoding the 26S rRNA sequence by using in vitro amplification,comprising a first amplification oligomer of up to 100 nucleotides inlength comprising a target binding region consisting of SEQ ID NO: 19, asecond amplification oligomer of up to 100 nucleotides in lengthcomprising a target binding region consisting of SEQ ID NO: 24, and adetection probe oligomer of up to 100 nucleotides in length comprising atarget binding region consisting of SEQ ID NO:
 28. 5. The composition ofclaim 4, further comprising at least one capture probe oligomer thatcontains a target binding region consisting of SEQ ID NO: 35 or 36,optionally with a 3′ tail sequence covalently attached to the targetspecific sequence.